Inclusion complexes of cyclodextrins with 4-amino-1,8-naphthalimides (part 2)

Fluorescence spectroscopy was used to characterize inclusion compounds between 4-amino-1,8-naphthalimides (ANI) derivatives and different cyclodextrins (CDs). The ANI derivatives employed were N-(12-aminododecyl)-4-amino-1,8-naphthalimide (mono-C₁₂ANI) and N,N′-(1,12-dodecanediyl)bis-4-amino-1,8-naphthalimide (bis-C₁₂ANI). The CDs used here were α-CD, β-CD, γ-CD, HP-α-CD, HP-β-CD and HP-γ-CD. The presence of CDs resulted in pronounced blue-shifts in the emission spectra of the ANI derivatives, with increases in emission intensity. This behavior was parallel to that observed for the dyes in apolar solvents, indicating that inclusion complexes were formed between the ANI and the CDs. Mono-C₁₂ANI formed inclusion complexes of 1:1 stoichiometry with all the CDs studied. Complexes with the larger CDs (HP-β-CD, HP-γ-CD and γ-CD) were formed by inclusion of the chromophoric ANI ring system, whereas the smaller CDs (α-CD, HP-α-CD and β-CD) formed complexes with mono-C₁₂ANI by inclusion of the dodecyl chain. Bis-C₁₂ANI formed inclusion complexes of 1:2 stoichiometry with HP-β-CD, HP-γ-CD and γ-CD, but did not form inclusion complexes with α-CD, HP-α-CD and β-CD. The data were treated in the case of the large CDs using a Benesi-Hildebrand like equation, giving the following equilibrium constants: mono-C₁₂ANI:HP-β-CD (K ₁₁ = 50 M^?1), mono-C₁₂ANI:HP-γ-CD (K ₁₁ = 180 M^?1), bis-C₁₂ANI:HP-β-CD (K ₁₂ = 146 M^?2), bis-C₁₂ANI:HP-γ-CD (K ₁₂ = 280 M^?2).     

Host–guest system of 4-nerolidylcatechol in 2-hydroxypropyl-β-cyclodextrin: preparation, characterization and molecular modeling

The interaction of 4-nerolidylcatechol (4-NRC), a potent antioxidant agent, and 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) was investigated by the solubility method using Fourier transform infrared (FTIR) methods in addition to UV–Vis, ¹H-nuclear magnetic resonance (NMR) spectroscopy and molecular modeling. The inclusion complexes were prepared using grinding, kneading and freeze-drying methods. According to phase solubility studies in water a B_S-type diagram was found, displaying a stoichiometry complexation of 2:1 (drug:host) and stability constant of 6494 ± 837 M^?1. Stoichiometry was established by the UV spectrophotometer using Job’s plot method and, also confirmed by molecular modeling. Data from ¹H-NMR, and FTIR, experiments also provided formation evidence of an inclusion complex between 4-NRC and HP-β-CD. 4-NRC complexation indeed led to higher drug solubility and stability which could probably be useful to improve its biological properties and make it available to oral administration and topical formulations.     

Complexation of ebastine with β-cyclodextrin derivatives

Complexation of ebastine (EB) with hydroxypropyl and methyl-β-cyclodextrin (HP-β-CD and Me-β-CD) was studied in aqueous solutions and in the solid state. The formation of inclusion complexes in aqueous solutions was analysed by the solubility method. The assays were designed using low CD concentrations compared with the solubility of these derivatives in order to avoid non-inclusion phenomena and to obtain a linear increase in EB solubility as a function of CD concentration. The values of complexation efficiency for HP-β-CD and Me-β-CD were 1.9 × 10^?2 and 2.1 × 10^?2, respectively. It seems that the non polar character of the methyl moiety slightly favoured complexation. In relation to solid state complexation, 1:1 EB:CD systems were prepared by kneading, and by heating a drug-CD mixture at 90 ºC. They were analysed using X ray diffraction analysis by comparison with their respective physical mixtures. A complex with a characteristic diffraction pattern similar to that of the channel structure of β-CD was formed with Me-β-CD in 1:1 melted and 1:2 EB:CD kneaded systems. Complexation with HP-β-CD was not clearly evidenced because only a slight reduction of drug crystallinity was detected. Finally, the loading of EB in two β-CD polymers cross-linked with epichlorohydrin yielded 7.3 and 7.7 mg of EB/g polymer respectively.     

Enrofloxacin inclusion complexes with cyclodextrins

Inclusion complexes using α-, β-, γ-, and hydroxypropyl-β-CD (HP-β-CD) were produced with the antibiotic enrofloxacin, with the aim of increasing its solubility by complexation. Phase solubility diagrams were obtained, to confirm the formation of inclusion complexes, and to determine the solubility enhancement and stability constant of each complex. Enrofloxacin inclusion in β-CD showed the highest value of the complex stability constant (35.56?mmol?L^?1), but the greatest increase in solubility was obtained using HP-β-CD reaching a 1258% increase over enrofloxacin solubility in the absence of CD. The order of highest enrofloxacin solubility achieved was: HP-β-CD?>?α-CD?>?γ-CD?>?β-CD. In addition, formation of complexes was confirmed by differential scanning calorimetry and thermogravimetry, applied to the complexes obtained by the kneading technique. The influence of citric acid, alone or as an adjunct of β-CD, on the solubility of enrofloxacin was also determined. A solution of 15?mmol?L^?1 citric acid dissolved 10?g?L^?1 of enrofloxacin, but a gradual increase in β-CD concentration in the presence of citric acid did not increase the degree of solubilization of enrofloxacin.     

Comparison between 2-hydroxypropyl-��-cyclodextrin and 2-hydroxypropyl-��-cyclodextrin for inclusion complex formation with danazol

Complexation in solution between danazol and two different cyclodextrins [2-hydroxypropyl-??-cyclodextrin (HP-??-CD) and 2-hydroxypropyl-??-cyclodextrin (HP-??-CD)] was studied using phase solubility analysis, and one- and two-dimensional ¹H-NMR. The increase of danazol solubility in the aqueous cyclodextrin solutions showed a linear relationship (A_L profile). The apparent stability constant, K _1:1, of each complex was calculated and found to be 51.7 × 10³ and 7.3 × 10³ M^?1 for danazol?CHP-??-CD and danazol?CHP-??-CD, respectively. ¹H-NMR spectroscopic analysis of varying ratios of danazol and the different cyclodextrins in a mixture of EtOD?CD₂O confirmed the 1:1 stoichiometry. Cross-peaks, from 2D ROESY ¹H-NMR spectra, between protons of danazol and H3?? and H5??of cyclodextrins, which stay inside the cyclodextrin cavity, proved the formation of an inclusion complex between danazol and the cyclodextrins. For HP-??-CD, the inclusion complex is formed by entrance of the isooxazole and the A rings of danazol in the cyclodextrin cavity. For HP-??-CD, two different inclusion structures may exist simultaneously in solution: one with the isooxazole and A ring in the cavity and the other with the C and D ring inside the cavity. DLS showed that self-aggregation of the CD??s was absent in the danazol HP-??-CD system up to a CD concentration of 10% and in the danazol HP-??-CD system up to a CD concentration of 5%.     

Study on the complexation of isoquercitrin with β-cyclodextrin and its derivatives by spectroscopy

The inclusion complexes of isoquercitrin (IQ) with cyclodextrins (CDs) including β-cyclodextrin (β-CD), hydroxypropyl-β-cyclodextrin (HP-β-CD) and dimethyl-β-cyclodextrin (DM-β-CD) have been investigated using the methods of steady-state fluorescence, UV-vis absorption and induced circular dichroism. The stoichiometric ratio of the three complexes was found to be 1:1 and the stability constants (K) were estimated from spectrofluorometric titrations, as well as the thermodynamic parameters. Maximum inclusion ability was measured in the case of DM-β-CD due to the increased hydrophobicity of the host cavity, followed by HP-β-CD and β-CD. The effect of pH on the complexation process was also quantitatively assessed. IQ exists in different molecular forms depending on pH and β-CDs were most suitable for inclusion of the neutral form of IQ. The phase-solubility diagrams obtained with β-CD, HP-β-CD and DM-β-CD were all classical A_L type. And DM-β-CD provided the best solubility enhancement, 12.3-fold increase compared to 2.8- and 7.5-fold increase for β-CD and HP-β-CD. The apparent stability constants obtained from the solubility data at 25 °C were comparable with those obtained from the fluorescence assays. Moreover, ¹H NMR was carried out, which revealed that the IQ favorably inserted into the inner cavity from the chromone part instead of the phenyl part, which was in agreement with molecular modeling studies.     

Thermo-reversible flurbiprofen liquid suppository with HP-β-CD as a solubility enhancer: improvement of rectal bioavailability

The purpose of this work was to develop a thermo-reversible flurbiprofen liquid suppository base composed of poloxamer and sodium alginate for the improvement of rectal bioavailability of flurbiprofen. Cyclodextrin derivatives such as α-, β-, γ-cyclodextrin and hydroxypropyl-β-cyclodextrin (HP-β-CD) were used to enhance the aqueous solubility of flurbiprofen. The effects of HP-β-CD and flurbiprofen on the physicochemical properties of liquid suppository were then investigated. Pharmacokinetic studies were performed after rectal administration of flurbiprofen liquid suppositories with and without HP-β-CD or after intravenous administration of commercial Lipfen^® (flurbiprofen axetil-loaded emulsion) to rats, and their pharmcokinetic parameters were compared. HP-β-CD decreased the gelation temperature and reinforced the gel strength and bioadhesive force of liquid suppository, while flurbiprofen was opposed to HP-β-CD. Thermo-reversible flurbiprofen liquid suppository showed the physicochemical properties suitable for rectal administration. The flurbiprofen liquid suppository with HP-β-CD showed significantly higher plasma levels, AUC and C_max of flurbiprofen than those of the liquid suppository without HP-β-CD, indicating that flurbiprofen could be well absorbed due to the enhanced solubility by formation of inclusion complex. Moreover, the flurbiprofen liquid suppository with HP-β-CD showed an excellent bioavailability in that the AUC of flurbiprofen after its rectal administration was not significantly different from that after intravenous administration of commercial Lipfen^®. It is concluded that HP-β-CD could be a preferable solubility enhancer for the development of liquid suppository containing poorly water-soluble drugs.     

Study on the inclusion interaction of ethyl violet with cyclodextrins by MWNTs/Nafion modified glassy carbon electrode

The interactions of ethyl violet (EV) with cyclodextrins (CDs) were investigated by Multi-wall carbon nanotubes/Nafion composite film modified glassy carbon electrode (MWNTs/Nafion/GCE). It was found that the MWNTs/Nafion composite film can effectively catalyze the electrode reaction of EV. The variation of the electrochemical behavior of EV upon the addition of CDs indicated the formation of the inclusion complexes of EV with β-CD, heptakis (2,3,6-tri-O-methyl)-β-CD (TM-β-CD), heptakis (2,6-di-O-methyl)-β-CD (DM-β-CD), hydroxypropyl-β-CD (HP-β-CD), and carboxymethyl-β-CD (CM-β-CD). The stoichiometry ratios of EV and the above five CDs were found to be 1:1. The inclusion ability obeyed the order: CM-β-CD > HP-β-CD > TM-β-CD > DM-β-CD > β-CD. The results showed that the modified β-CDs exhibited stronger binding ability than native β-CD, especially the charged CM-β-CD, which implied that the inclusion capacity depends on not only size matching and hydrophobicity but also electrostatic interaction. ¹HNMR spectra and molecule mechanics calculations suggested that EV was included into the cavity of β-CD from the wider side.     

Preparation, characterization and pharmacodynamic activity of supramolecular and colloidal systems of rosuvastatin–cyclodextrin complexes

In the present study influence of nature of selected cyclodextrins (CDs) and of methods of preparation of drug–CD complexes on the oral bioavailability, in vitro dissolution studies and pharmacodynamic activity of a sparingly water soluble drug rosuvastatin (RVS) was investigated. Phase solubility studies were conducted to find the interaction of RVS with β-CD and its derivatives, which indicated the formation of 1:1 stoichiometric inclusion complex. The apparent stability constant (K_1:1) calculated from phase solubility diagram were in the rank order of β-CD < hydroxypropyl-β-cyclodextrin (HP-β-CD) < randomly methylated-β-cyclodextrin (RM-β-CD). Equimolar drug–CD solid complexes prepared by different methods were characterized by the Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC) and X-ray diffractometry (XRD). FTIR study demonstrated the presence of intermolecular hydrogen bonds and ordering of the molecule between RVS and CDs in inclusion complexes. DSC and XRD analysis confirmed formation of inclusion complex by freeze dried method with HP-β-CD and RM-β-CD. Aqueous solubility and dissolution studies indicated improved dissolution rates of prepared complexes in comparison with drug alone. Moreover, CD complexes demonstrated of significant improvement in reducing total cholesterol and triglycerides levels as compared to pure drug. However the in vivo results only partially agreed with those obtained from phase solubility studies.     

Synthesis and structural characterization of 18-, 19-, 20- and 22-membered Schiff base macrocycles

The complexation parameters of dipyridamole (Dipy) with β-cyclodextrin (β-CD) were investigated by using several techniques including phase solubility diagrams (PSD), proton nuclear magnetic resonance (¹H-NMR), x-ray powder diffractometry (XRPD), differential scanning calorimetry (DSC), scanning electron microscopy (SEM) and molecular mechanical modeling (MM⁺). From the pH-solubility profiles, two basic pK _as at 6.4 and 2.7 were estimated. The linear correlation of the free energy of Dipy/β-CD complex formation (ΔG ₁₁) with the corresponding free energy of inherent Dipy aqueous solubility (ΔG _So), obtained from the linear variation of ln K ₁₁ with that of the inherent Dipy solubility (ln S _o) at different pHs and ionic strengths, was used to measure the contribution of the hydrophobic character of Dipy to include into the hydrophobic β-CD cavity. Complex formation of Dipy was driven by favorable enthalpy (ΔH° = ?14.8 kJ/mol) and entropy (ΔS° = 31.9 J/mol K) factors. ¹H-NMR and molecular mechanical modeling studies indicate the formation of different isomeric 1:1 and 1:2 complexes, where both the piperidine and diethanolamine moieties get separately included into the β-CD cavity. Molecular mechanical modeling computations indicate that the dominant driving force for complexation is Van der Waals with lower contribution from electrostatic interactions. ¹H-NMR and XRPD, DSC, SEM studies of isolated solid complexes indicate the formation of inclusion complexes in aqueous solution.     

Inclusion complexes of Sulconazole with β-cyclodextrin and hydroxypropyl β-cyclodextrin: characterization in aqueous solution and in solid state

Complexation between sulconazole (SULC), an imidazole derivative with in vitro antifungal and antiyeast activity, and β-cyclodextrins (β-CD and HP-β-CD) was studied in solution and in solid states. Complexation in solution was evaluated using solubility studies and nuclear magnetic resonance spectroscopy (¹H-NMR). In the solid state, differential scanning calorimetry (DSC), thermal gravimetric analysis (TGA), scanning electron microscopy (SEM) and RX diffraction studies were used. Solubility studies suggested the existence of inclusion complex between SULC and β-CD or HP-β-CD. ¹H-NMR spectroscopy studies showed that the complex formed occurs by complexation of imidazole ring into inner cavity. DSC studies showed the existence of a complex of SULC with β-CD. The TGA and RX studies confirmed the DSC results of the complex. Solubility of SULC in solid complexes was studied by the dissolution method and it was found to be much more soluble than the uncomplexed drug.     

Competitive Binding of Tolmetin to β-Cyclodextrin and Human Serum Albumin: <Superscript>1</Superscript>H NMR and Fluorescence Spectroscopy Studies

The host–guest interaction of tolmetin (TOL) with β-cyclodextrin (β-CD) and the influence of human serum albumin (HSA) on the formation of the inclusion complex were studied by 1D and 2D NMR spectroscopy. The TOL/β-CD inclusion complex formed at a molar ratio of 1:1 with a binding constant value of 2164.5 L·mol^?1. Data analysis showed that the addition of 10 μmol·L^?1 of HSA weakened the strength of TOL binding to β-CD (K _a = 1493 L·mol^?1). The interaction of TOL with HSA in the absence and presence of β-CD was studied by analyzing the fluorescence quenching data. The Stern–Volmer quenching constants and the binding constants are found to be smaller in the presence of β-CD, suggesting that β-CD hinders the strong interaction of TOL with HSA by complex formation. Additionally, the presence of β-CD does not induce conformational and microenvironmental changes on HSA.     

Inclusion complexes of rosmarinic acid and cyclodextrins: stoichiometry,association constants,and antioxidant potential

The interaction between β-cyclodextrin (β-CD) and the polyphenol rosmarinic acid (RA) is here reported by ¹H NMR titration experiments. The formation of an aqueous soluble inclusion complex is confirmed and valuable information regarding mode of penetration of guest into β-CD, stoichiometry, and stability of the complex is obtained. The analysis by the continuous variation method shows the undoubted formation of 1:1 β-CD/RA complex. Additionally, the estimated apparent association constants reveal the importance of the asymmetry of the RA in the complexation; the incorporation of the catechol moiety closer to the carboxylic group is more favorable (K?=?2,028 M^?1) than from the other end of the RA molecule (K?=?1,184 M^?1). Finally, we have also investigated the antioxidant activity and storage stability of the β-CD/RA complexed system; the presence of β-CD was found to produce a remarkable enhancement on the antioxidant activity.     

CE, with Hydroxypropyl-β-Cyclodextrin as Chiral Selector, for Separation and Determination of the Enantiomers of Amlodipine in the Serum of Hypertension Patients

A capillary electrophoretic method for separation of the enantiomers of amlodipine in the serum of hypertension patients has been established and validated. The two enantiomers were separated in a fused-silica capillary with phosphate running buffer (75 mmol L^?1, pH 2.5) containing 15 mmol L^?1 hydroxypropyl-β-cyclodextrin (HP-β-CD). The effects on the separation of buffer pH and concentration, separation potential, and concentration of HP-β-CD were investigated. The range of quantitation for both enantiomers was 2.0–16.0 μg mL^?1. Intra-day and inter-day relative standard deviation (RSD; n = 5) was <10%. The limits of detection (LOD) and quantification (LOQ) of the amlodipine enantiomers, at 214 nm, were approximately 0.5 and 0.7 μg mL^?1, respectively (S/N = 3 and 10, respectively; 5-s injection). Recovery was always >85%. Results from enantiomer separation and quantification showed that concentrations of the enantiomers of amlodipine in serum from an elderly patient were higher than in serum from a young patient administered the same dose. The method was useful for determining the concentration of the enantiomers of amlodipine in hypertension patient serum and for monitoring the transition behavior of the enantiomers in humans. The method proved suitable for application to the separation of the enantiomers of amlodipine and analysis of clinical samples.     

Kinetic study on reactive extraction for chiral separation of phenylsuccinic acid enantiomers

The kinetics of the extraction of phenylsuccinic acid (PSA) enantiomers by hydroxypropyl-β-cyclodextrin (HP-β-CD) in a modified Lewis cell was studied, in which HP-β-CD dissolved in 0.1 mol L^?1 NaH₂PO₄/H₃PO₄ buffer solution (pH = 2.5) was selected as the chiral extractant. PSA enantiomers were extracted from organic phase to aqueous phase in the extraction module. The theory of extraction accompanied by a chemical reaction has been used to obtain the intrinsic kinetics of this extraction module. The different parameters affecting the extraction rate such as agitation speed, interfacial area, initial concentration of PSA enantiomers in organic phase as well as HP-β-CD concentration in aqueous phase were separately studied. The experimental results demonstrate that the extraction reactions are fast. The reactions were found to be first order with respect to PSA and second order with respect to HP-β-CD with forward rate constants of 3.4 × 10^?2 m⁶ mol^?2 s^?1 for R-PSA and 9.96 × 10^?3 m⁶ mol^?2 s^?1 for S-PSA. These data will be useful in the design of extraction processes.     

Water-insoluble β-cyclodextrin polymer crosslinked by citric acid: synthesis and adsorption properties toward phenol and methylene blue

Water-insoluble β-cyclodextrin polymer (β-CDP) crosslinked by citric acid was obtained with a yield of 65% through an environment friendly synthesis procedure. FT-IR spectra disclosed that the hydroxyl groups of β-CD had reacted and condensated with the carboxyl groups of citric acid, and at the same time the structural characteristics of β-CD were essentially maintained in β-CDP. The β-CDP exhibited notable adsorption capability toward phenol (q _max = 13.8 mg g^?1) and especially large adsorption capability toward methylene blue (q _max = 105 mg g^?1). The concentration of methylene blue in water could be reduced to 0.11 mg L^?1 by the β-CDP, indicating the excellent adsorption sensitivity of β-CDP toward methylene blue. The adsorption results disclosed that the interior cavity and inclusion property of β-CD were maintained in the synthesized β-CDP.     

Inclusion complexes of lamotrigine and hydroxy propyl β-cyclodextrin: solid state characterization and dissolution studies

Lamotrigine (LMN) is an antiepileptic drug, with poor aqueous solubility, which might lead to erratic bioavailability. The objective of the present work was to improve the dissolution characteristics of the LMN using Hydroxy propyl β-cyclodextrin (HP β-CD), which might offer reliable bioavailability. The phase solubility profile was classified as A _L -type, revealing 1:1 stoichiometric complexation, with a stability constant (Ks) of 573 M^?1. Binary systems of LMN and HP β-CD were prepared in different molar ratios (1:1, 1:2, 1:3 and 1:4) by kneading method. The binary systems were characterized by Fourier Transform Infrared (FT-IR) Spectroscopy, Differential Scanning Calorimetry (DSC) and Powder X-ray Diffraction Analysis (PXRD). Results revealed that in the kneaded products the entire drug was entrapped inside the HP β-CD cavity and reduction in drug crystallinity also took place, which may be responsible for improved dissolution characteristics as compared to that of the pure drug as depicted from the dissolution studies.     

Destabilization of cytochrome c by modified β-cyclodextrin

To clarify the effect of cyclodextrin (CD) on the stability of cytochrome c, the thermal denaturation of cytochrome c was measured by differential scanning calorimetry in aqueous solutions of β-CD modified with three substituents: methyl, acetyl, and 2-hydroxylpropyl groups. The midpoint temperature of thermal denaturation decreased with the addition of modified β-CDs, indicating that cytochrome c was destabilized. The destabilization effect of CD depended on the substituent and increased in the order of acetyl>methyl>2-hydroxypropyl. The estimated binding number and binding constant of the modified β-CDs for cytochrome c are 5.0 ± 1.0 and 10.3 ± 2.9 M^?1 for methyl-β-CD, 13.8 ± 3.6 and 4.7 ± 1.6 M^?1 for acetyl-β-CD, and 2.8 ± 0.9 and 7.0 ± 3.0 M^?1 for 2-hydroxypropyl-β-CD. The destabilization effect of acetyl-β-CD is the highest because many CD molecules interact with proteins by the inclusion effect of CD and the inhibition effect of the acetyl group on the hydrogen bond in the secondary structure. In contrast, the stabilization effect of 2-hydroxypropyl-β-CD is the smallest because the steric exclusion of the 2-hydroxypropyl group inhibits the binding of CD to cytochrome c as compared with the smaller structure of the methyl group. Dependency of the destabilization effect on the molar ratio of CD to cytochrome c suggests that the destabilization effect of CD is caused not only by the “direct” interaction of CD with proteins but also by the “indirect” interaction of CD which promotes the solvation of hydrophobic groups by altering the water structure as observed in urea.     

Effect of hydroxypropyl-β-cyclodextrin complexation on the aqueous solubility, structure, thermal stability, antioxidant activity, and tyrosinase inhibition of paeonol

The objective of this paper is to study the effect of hydroxypropyl-β-cyclodextrin (HP-β-CD) complexation on the aqueous solubility, structure, thermal stability, antioxidant activity, and tyrosinase inhibition of paeonol (PAE). The inclusion complex (PAE-HP-β-CD complex) of HP-β-CD and PAE was prepared by a freeze-drying method. Phase solubility tests showed that the stability constant of the inclusion complex was about 33.8?M^?1 at 25?°C. The experimental results of proton nuclear magnetic resonance (H-NMR) spectroscopy, differential scanning calorimetry (DSC) and X-ray diffraction (XRD) suggested that PAE was included by HP-β-CD to form the PAE-HP-β-CD complex. Furthermore, the thermogravimetric analysis (TGA) results showed that the thermal stability of PAE was improved when it was complexed with HP-β-CD. Comparing the antioxidant activity of PAE with that of the PAE-HP-β-CD complex at the same concentration revealed that the complex of PAE with HP-β-CD was better able to eliminate radical. Furthermore, the experimental results revealed that the formation of a complex with HP-β-CD increased the water solubility of PAE, improving its apparent inhibitive activity of tyrosinase.